This invention relates to the microbial production, via recombinant DNA technology, of human leukocyte interferons for use in thie treatment of viral and neoplastic diseases, and to the means and end products of such production.
The publications and other materials referred to herein to illuminate the background of the invention and, in particular cases, to provide additional detail respecting its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.
Leukocyte Interferon
Human leukocyte interferon was first discovered and prepared in the form of very crude precipitates by Isaacs and Lindenmann (3). Efforts to purify and characterize the material have been ongoing since that time, and have led to the preparation of relatively homogeneous leukocyte interferons derived from normal or leukemic (chronic myelogenous leukemia or xe2x80x9cCMLxe2x80x9d) donors"" leukocytes (4). These interferons are a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells (1,2). In addition, interferon can act to inhibit cell proliferation and modulate immune response. These properties have prompted the clinical use of leukocyte interferon as a therapeutic agent for the treatment of viral infections and malignancies.
Leukocyte interferons have been purified to essential homogeneity (7,8), and reported molecular weights range from about 17,500 to about 21,000. The specific activity of these preparations is remarkably high, 2xc3x97108 to 1xc3x97109 units/mg protein, but yields from cell culture methods have been discouragingly low. Nevertheless, advances in protein sequencing techniques have, in our hands, permitted the determination of partial amino acid sequences (4). Elucidation of the glycosylation of various leukocyte interferons is not at present complete, but it is now clear (by virtue of the work reported infra) that differences in glycosylation among family members does not alone account for the spectrum of molecular weights observed. Instead, the leukocyte interferons differ markedly in amino acid composition and sequence, and amino acid homology is, in some cases, less than 80 percent.
While isolation from donor leukocytes has provided sufficient material for partial characterization and limited clinical studies with homogeneous leukocyte interferon, it is a totally inadequate source for the amounts of interferon needed for large scale clinical trials and for broad scale prophylactic and/or therapeutic use thereafter. Indeed, presently clinical investigations employing human leukocyte-derived interferons in antitumor and antiviral testing have principally been confined to crude ( less than 1 percent pure) preparations of the material, and long lead times for the manufacture of sufficient quantities, even at unrealistic price levels, have critically delayed investigation on an expanded front.
Recombinant DNA Technology
With the advent of recombinant DNA technology, the controlled microbial production of an enormous variety of useful polypeptides has become possible. Already in hand are bacteria modified by this technology to permit the production of such polypeptide products such as somatostatin (5), the (component) A and B chains of human insulin (9) and human growth hormone (18). More recently, recombinant DNA techniques have been used to occasion the bacterial production of proinsulin and thymosin alpha 1, an immune potentiating substance produced by the thymus.
Other workers have reported on the obtention of DNA coding for human leukocyte interferon and to resultant proteins having leukocyte interferon activityxe2x80x94cf. Nagata et al., Nature 284, 316 (1980); Mantei et al., Gene 10, 1 (1980). See also Taniguchi et al., Nature 285, 547 (1980).
The workhorse of recombinant DNA technology is the plasmid, a non-chromosomal loop of double-stranded DNA found in bacteria and other microbes, oftentimes in multiple copies per cell. Included in the information encoded in the plasmid DNA is that required to reproduce the plasmid in daughter cells (i.e., a xe2x80x9crepliconxe2x80x9d) and ordinarily, one or more selection characteristics such as, in the case of bacteria, resistance to antibiotics which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or xe2x80x9crestriction enzymexe2x80x9d, each of which recognizes a different site on the plasmidic DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent to the cleavage site. DNA recombination is performed outside the cell, but the resulting xe2x80x9crecombinantxe2x80x9d plasmid can be introduced into it by a process known as transformation and large quantities of the heterologous gene-containing recombinant plasmid obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DNA message, the resulting expression vehicle can be used to actually produce the polypeptide sequence for which the inserted gene codes, a process referred to as expression.
Expression is initiated in a region known as the promoter which is recognized by and bound by RNA polymerase. In some cases, as in the tryptophan or xe2x80x9ctrpxe2x80x9d promoter preferred in the practice of the present invention, promoter regions are overlapped by xe2x80x9coperatorxe2x80x9d regions to form a combined promoter-operator. Operators are DNA sequences which are recognized by so-called repressor proteins which serve to regulate the frequency of transcription initiation at a particular promoter. The polymerase travels along the DNA, transcribing the information contained in the coding strand from its 5xe2x80x2 to 3xe2x80x2 end into messenger RNA which is in turn translated into a polypeptide having the amino acid sequence for which the DNA codes. Each amino acid is encoded by a nucleotide triplet or xe2x80x9ccodonxe2x80x9d within what may for present purposes be referred to as the xe2x80x9cstructural genexe2x80x9d, i.e. that part which encodes the amino acid sequence of the expressed product. After binding to the promoter, the RNA polymerase first transcribes nucleotides encoding a ribosome binding site, then a translation initiation or xe2x80x9cstartxe2x80x9d signal (ordinarily ATG, which in the resulting messenger RNA becomes AUG), then the nucleotide codons within the structural gene itself. So-called stop codons are transcribed at the end of the structural gene whereafter the polymerase may form an additional sequence of messenger RNA which, because of the presence of the stop signal, will remain untranslated by the ribosomes. Ribosomes bind to the binding site provided on the messenger RNA, in bacteria ordinarily as the mRNA is being formed, and themselves produce the encoded polypeptide, beginning at the translation start signal and ending at the previously mentioned stop signal. The desired product is produced if the sequences encoding the ribosome binding site are positioned properly with respect to the AUG initiator codon and if all remaining codons follow the initiator codon in phase. The resulting product may be obtained by lysing the host cell and recovering the product by appropriate purification from other bacterial protein.
We perceived that application of recombinant DNA technology would be the most effective way of providing large quantities of leukocyte interferon which, despite the absence in material so produced of the glycosylation characteristic of human-derived material, could be employed clinically in the treatment of a wide range of viral and neoplastic diseases.
More particularly, we proposed and have since succeeded in producing mature human leukocyte interferon microbially, by constructing one or more genes therefor which could then be inserted in microbial expression vehicles and expressed under the control of microbial gene regulatory controls.
Our approach to obtaining a first leukocyte gene involved the following tasks:
1. Partial amino acid sequences would be obtained by characterization of leukocyte interferon purified to essential homogeneity, and construct sets of synthetic DNA probes constructed whose codons would, in the aggregate, represent all the possible combinations capable of encoding the partial amino acid sequences.
2. Bacterial colony banks would be prepared containing cDNA from induced messenger RNA. Other induced mRNA that had been radio-labelled would be hybridized to plasmid cDNA from this bank. Hybridizing mRNA would be eluted and tested for translation into interferon in oocyte assay. Plasmid DNA from colonies shown positive for interferon in this manner would be further tested for hybridization to probes made as described in (1) above.
3. Parallel to the approach in part (2) above, induced mRNA-derived cDNA in plasmids would be used to form an independent bank of transformant colonies. The probes of part (1) would be used to prime the synthesis of radio-labelled single stranded cDNA for use as hybridization probes. The synthetic probes would hybridize with induced mRNA as template and be extended by reverse transcription to form induced, radio-labelled cDNA. Clones from the colony bank that hybridized to radio-labelled cDNA obtained in this manner would be investigated further to confirm the presence of a full-length interferon encoding gene. Any partial length putative gene fragment obtained in parts (1) or (2) would itself be used as a probe for the full-length gene.
4. The full-length gene obtained above would be tailored, using synthetic DNA, to eliminate any leader sequence that might prevent microbial expression of the mature polypeptide and to permit appropriate positioning in an expression vehicle relative to start signals and the ribosome binding site of a microbial promoter. Expressed interferon would be purified to a point permitting confirmation of its character and determination of its activity notwithstanding the absence of glycosylation.
5. The interferon gene fragment prepared in the foregoing fashion could itself be used in probing, by hybridization, for other partially homologous leukocyte interferon species.
We have discovered and, through recombinant DNA technology, enabled the microbial production in high yield of the family of homologous leukocyte interferons (sans glycosylation) as mature polypeptides, essentially unaccompanied by the corresponding presequence or any portion thereof. These may be directly expressed, recovered and purified to levels fitting them for use in the treatment of viral and malignant diseases of animals and man. Family members so far expressed have proven efficacious in in vitro testing and, in the first such demonstration of its kind, in in vivo testing as well, the latter involving the first mature leukocyte interferon to have been microbially produced. The invention comprises the interferons so produced and means of producing them.
Reference herein to the expression of a xe2x80x9cmature leukocyte interferon,xe2x80x9d connotes the bacterial or other microbial production of an interferon molecule unaccompanied by associated glycosylation and the presequence that (as we have discovered) immediately attends mRNA translation of a human leukocyte interferon genome. Mature leukocyte interferon, according to the present invention, is immediately expressed from a translation start signal (ATG) just before the first amino acid codon of the natural product, in which event the mature polypeptide includes the methionine for which ATG codes without essentially altering its character, or the microbial host may process the translation product to delete the initial methionine. Mature leukocyte interferon could be expressed together with a conjugated protein other than the conventional leader, the conjugate being specifically cleavable in an intra- or extracellular environment. See British Patent Publication No. 2007676A. Finally, the mature interferon could be produced in conjunction with a microbial xe2x80x9csignalxe2x80x9d peptide which transports the conjugate to the cell wall, where the signal is processed away and the mature polypeptide secreted.
Particular leukocyte interferon proteins hereof have been defined by means of determined DNA gene and deductive amino acid sequencingxe2x80x94cf. FIGS. 3, 4, 8 and 9, for example. It will be understood that for these particular interferons, indeed all of the family of leukocyte interferon proteins embraced herein, natural allelic variations exist and occur from individual to individual. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. For each leukocyte interferon protein hereof, labelled LeIF A, LeIF B . . . LeIF J, etc., such allelic variations are included within the scope of the label or term defining such, and thus, this invention.